CLINICAL ASPECTS OF

BIOCHEMISTRY
ENZYMES IN MEDICAL
DIAGNOSIS

Raymond Oliver A. Cruz, MD

raymondolivercruz.wordpress.com
Steps in the Investigation of a Patient

• Patient History
• Physical Examination
• Laboratory Tests
• Imaging Techniques
• Diagnosis
• Therapy
• Evaluation
 ENZYMES IN CLINICAL
DIAGNOSIS
• Alteration in plasma enzyme levels can
lead to diseases
• Plasma enzyme levels can be used as
diagnostic tools
 Blood Composition
Plasma is fluid component of blood.
Comprises ~55% of total volume of
whole blood. Contains proteins, sugars,
Plasma vitamins,minerals, lipids, lipoproteins and
clotting factors.
95% of plasma is water

White Blood cells (WBC)

& Platelets Cellular
Red Blood cells (RBC) Components

Whole Blood Whole Blood after centrifugation

Note: clotting has been prevented
 Blood Composition
If blood is collected and allowed to stand it will clot. If blood
is then centrifuged the fluid portion is
known as SERUM
Plasma is fluid component of blood.
Comprises ~55% of total volume of
whole blood. Contains proteins, sugars,
Serum vitamins,minerals, lipids, lipoproteins
No clotting factors
95% of plasma is water

Blood Clot
-comprised of clotting factors (Fibrin,platets etc)
-RBCs

Whole Blood Whole Blood after clotting and centrifugation

 Collection Tubes
(Vacutainers)

Separator Gel

Serum
Separator Gel

Serum Separator Tube (SST) Clot

 Collection tubes

• Red-top tubes contain no

anticoagulants or preservatives
• Red-top tubes are used for collecting
serum
– 10-15 minutes is required to allow blood to
clot before centrifuging
– Used for blood bank specimens, some
chemistries
 Collection tubes

• Gold (and “tiger”) top tubes contain a

gel that forms a physical barrier
between the serum and cells after
centrifugation
• No other additives are present
• Gel barrier may affect some lab tests
 Collection tubes

• Used for Glucose measurement.

• After blood collection, glucose concentration
decreases significantly because of cellular
metabolism
• Gray-top tubes contain either:
– Sodium fluoride and potassium oxalate, or
– Sodium iodoacetate
• Both preservatives stabilize glucose in plasma by
inhibiting enzymes of the glycolytic pathway
– NaF/oxalate inhibits enolase
– Iodoacetate inhibits glucose-3-phosphate dehydrogenase
 Collection tubes

• Green-top tubes contain either the Na, K, or lithium

(Li) salt of heparin. Most widely used anticoagulant
for chemistry tests.
– Should not be used for Na, K or Li measurement
– Can effect the size and integrity of cellular blood
components and not recommended for hematology studies
• Heparin accelerates the action of antithrombin III,
which inhibits thrombin, so blood does not clot
(plasma)
• The advantage of plasma is that no time is wasted
waiting for the specimen to clot
 Collection tubes

• Lavender-top tubes contain the K salt of

ethylenediaminetetraacetic acid
(EDTA), which chelates calcium
(essential for clot formation) and inhibits
coagulation
• Used for hematology, and some
chemistries
• Cannot be used for K or Ca tests
 Collection tubes

• Blue-top tubes contain sodium citrate,

which chelates calcium and inhibits
coagulation
• Used for coagulation studies because it
is easily reversible.
 Collection tubes

• Brown and Royal Blue top tubes are

specially cleaned for trace metal studies
– Brown-top tubes are used for lead (Pb)
analysis
– Royal blue-top tubes are used for other
trace element studies (acid washed)
 TRANSAMINASES
A. ASPARTATE AMINOTRANSFERASE
(AST) OR SERUM GLUTAMIC
OXALOACETIC TRANSAMINASES
(SGOT OR GOT)
- catalyzes the reversible transfer of an
amino group from glutamic acid to
oxaloacetic acid and from aspartate to
alpha-ketoglutarate
 AST / SGOT
• Pyridoxal phosphate acts as coenzyme
• increased concentration in cardiac
muscles, skeletal muscles, and liver
• Normal value: 0-35 IU/L
MYOCARDIAL INFARCTION
• Elevated SGOT when the enzyme is
released from necrotic cardiac tissue
• 6-12 hrs after estimated coronary
occlusion, SGOT increases to more
than 40 IU/L
• The peak value is reached after 24-48
hrs (110-240 IU/L)
MYOCARDIAL INFARCTION
• On the 4th-7th day, SGOT levels return
to normal
• However, this test is no longer indicated
for the diagnosis of myocardial
infarction
 MUSCULAR DYSTROPHY
• Degenerative disease of the muscles
• SGOT leaks out of the muscle during
the process of degeneration
 SGOT is also increased in:
• Acute viral hepatitis, biliary tract
obstruction, alcoholic hepatitis and
cirrhosis, liver infection, liver cancer
• SGOT is decreased in Vitamin B6
(Pyridoxine) deficiency
 ALANINE
AMINOTRANSFERASE (ALT)
• Also called serum glutamic-pyruvic
transaminase (GPT or SGPT)
• catalyzes the reversible transfer of an
amino group from glutamic acid to
pyruvic acid and from alanine to alpha-
ketoglutarate
 ALANINE
AMINOTRANSFERASE (ALT)
• Pyridoxal phosphate acts as coenzyme
• Increased concentration in liver and
kidneys
• Elevated in viral hepatitis and other
forms of liver disease
• In the liver, GPT activity is higher than
GOT
 LACTIC ACID
DEHYDROGENASE (LDH)
• Catalyzes the reversible oxidation of
lactate to pyruvate with NAD as the
hydrogen acceptor
• has 5 isoenzymes which contains 4
polypeptide chains
HHHH HHHM HHMM HMMM
MMMM (predominates in muscles)
 LACTIC ACID
DEHYDROGENASE (LDH)
• Normal value is from 55-140 IU/L
• Increased concentration in the liver,
heart, kidneys, skeletal muscles, and
whole blood
• In MI, serum levels begin to rise after
48-72 hours after onset of pain
 LACTIC ACID
DEHYDROGENASE (LDH)
• In liver disease, elevations are of
smaller degree compared to elevation of
transaminases
• In cancer, LDH increases, particularly in
abdominal and lung cancers
• LDH from effusions in cancer sites are
useful for diagnosis
 PHOSPHATASES
• A phosphatase is an enzyme that
dephosphorylates its substrate; i.e., it
hydrolyzes phosphoric acid monoesters into a
phosphate ion and a molecule with a free
hydroxyl group
• This action is directly opposite to that of
phosphorylases and kinases, which attach
phosphate groups to their substrates by using
energetic molecules like ATP
Riboflavin-5-PO4
 PHOSPHATASES
• Include alkaline and acid phosphatases
• hydrolyzes a wide variety of organic
phophates
 ALKALINE PHOSPHATASE
• Found in liver, bone, intestine, placenta
• normal value: 41-133 IU/L
• optimal activity at pH 9.8
• excreted through the biliary system
 ALKALINE PHOSPHATASE
• Increased in obstructive hepatobiliary
disease, bone disease or bone cancer,
hyperparathyroidism, third trimester of
pregnancy, gastrointestinal disease
• Decreased in hypophosphatemia
• Normal in osteoporosis
 ALKALINE PHOSPHATASE
• In obstructive jaundice, blockage of bile
ducts results in elevated levels
• In Paget’s disease, increased alkaline
phosphatase results from increased
activity of osteoblastic cells, as they try
to rebuild bone dissolved uncontrollably
by osteoclasts
 ACID PHOSPHATASE
• Increased concentration in serum,
prostate, semen, and liver
• Optimal activity at pH 4.9-5.0
• Increased levels of prostatic acid
phosphatase in sera of males with
metastasis
 ACID PHOSPHATASE
• Human prostatic acid phosphatase
(hPAP) is a glycoprotein synthesized in
the epithelial cells of the prostate gland
 ACID PHOSPHATASE
• However, hPAP is not very useful as a
diagnostic test for prostate cancer
• Increased concentration in semen
makes it useful in forensic medicine in
investigations of rape and similar
offenses
 ACID PHOSPHATASE
• Increased elevation in total acid
phosphatase is encountered in women
with metastatic breast cancer,
metastasis being the source of the
enzyme
 AMYLASE
• Hydrolase which catalyzes the
hydrolytic splitting of the alpha-1,4
linkages of polysaccharides such as
starch and glycogen
• Beta amylases in bacteria are
exoamylases which split off 2 glucose
units at a time
• Normal value: 20-110 U/L
 AMYLASE
• Alpha amylase in humans are
endoamylases
• Increased concentration in the pancreas
(acinar cells) and salivary glands, and
and inflammation or obstruction of these
organs increase serum levels
(pancreatitis, mumps)
 AMYLASE
• However, amylase is nonspecific
because it is also produced by the GIT,
Ovary, and skeletal muscle
• Elevated in acute pancreatitis but
normal or low in chronic pancreatitis
 LIPASE
• Hydrolyzes the glycerol esters of long-
chain fatty acids
• Normal value: 0-160 U/L
• Produced in the liver, intestine, tongue,
stomach, and other cells
• Increased serum levels in pancreatic
disorders, hepatic disease, diabetes,
GIT disorders
 LIPASE
• Sensitivity of lipase in acute pancreatitis
is similar to that of amylase, but serum
lipase remains elevated longer than
amylase
• The specificity of amylase and lipase for
diagnosing acute pancreatitis is poor
 CREATINE
PHOSPHOKINASE (CPK)
• Also called creatine kinase (CK)
• catalyzes the reversible phosphorylation
of creatine by ATP
• Normal value: 32-267 IU/L
• The brain, heart, and skeletal muscles
are rich in CPK, and these organs
secrete this enzyme when damaged
 CREATINE
PHOSPHOKINASE (CPK)
• Consists of 3 isoenzymes made up of 2
subunits, M and B
• The fraction with the greatest
electrophoretic mobility is CK1(BB);
CK2 (MB) is intermediate and CK3
(MM) moves slowest towards the anode
 CREATINE
PHOSPHOKINASE (CPK)
• Skeletal muscle is characterized by
isoenzyme MM and brain by isoenzyme
BB
• Myocardium has approximately 40%
MB isoenzyme, and CKMB levels
increase 3 to 5 hours after myocardial
infarction, which peaks after 24 hrs and
persists for 2-3 days
 RENAL FUNCTION TESTS
Kidney functions:
• maintains the body’s internal
environment
• excretion of waste products from the
blood
• selective filter to reabsorb water and
preserve essential solutes
• Normal kidney function depends upon
an adequate flow of blood to the organ
Any situation that interferes with the renal
supply will result in:
1. Diminished kidney function
2. Altered amount and composition of
urine
3. Accumulation of metabolic products in
blood
RENAL CLEARANCE TESTS
• Useful to monitor kidney’s excretory
function
• measures the amount of the substance
excreted in the urine as compared to
the concentration of the same
substance in the plasma
 CREATININE CLEARANCE
TEST
• Creatinine is an anhydride of creatine
• It is produced after creatine phosphate
has donated its phosphate group to
ATP for the replenishment of the energy
reserve
• This test is a relatively accurate and
useful measure of glomerular filtration
rate
 CREATININE CLEARANCE
TEST
• Fluid intake and excretion do not affect
creatinine clearance
• This test is theoretically reliable, but
often compromised by incomplete urine
collection
• Calculated from measurement of the
urine creatinine, plasma creatinine, and
urine flow rate
 CREATININE CLEARANCE
TEST
• Increased in high cardiac output states,
exercise, cardiomegaly, diabetes
mellitus, infections, and hypothyroidism
• Decreased in renal failure and
decreased renal blood flow states
• Normal value: 90-130 mL/min
 SERUM
CREATINE/CREATININE
• Removed from the plasma by
glomerular filtration and excreted in the
urine without being reabsorbed by the
tubules
• Do not increase until renal function is
greatly impaired
• Increased muscle destruction states
increases its levels
 SERUM
CREATINE/CREATININE
• For each 50% reduction in glomerular
filtration rate, serum creatinine
approximately doubles
• Normal: 0.6-1.2 mg/dL
• Increased in renal failure, urinary
obstruction, hypothyroidism
• Decreased in reduced muscle mass
 UREA NITROGEN (BUN)
• Urea is synthesized in the liver from ammonia
through enzymes of the urea cycle
• Normal value: 8-20 mg/dL
• This test is frequently requested along along
with serum creatinine since simultaneous
determination aids in the differential diagnosis
of prerenal, renal and postrenal hypovolemia
(normal ratio is 12:1-20:1)
CAUSES OF HYPERURICEMIA
• Prerenal causes:
volume contraction, hypotension, heart
failure, liver failure
• Renal causes:
Acute glomerulonephritis, polycystic kidney
disease, tubular necrosis
• Postrenal causes:
Ureteral obstruction, bladder outlet
obstruction
 URIC ACID (BUA)
• Waste product from purines of the diet and
those synthesized by the body
• End product of purine catabolism in man
• Normally about ½ of the total uric acid is
eliminated and replaced daily
• Most helpful in the diagnosis of gout
(increased in patients with gout)
• Normal value: males 2.4-7.4 mg/dL
females 1.4-5.8 mg/dL
 URIC ACID (BUA)
• An increase in serum uric acid concentration
occurs with increased nucleoprotein
synthesis or catabolism (blood dyscrasias,
therapy of leukemia) or decreased renal uric
acid excretion (thiazide diuretic therapy or
renal failure)
• Xanthine oxidase inhibitors (allopurinol)
decrease its levels
 URIC ACID (BUA)
• Uric acid of urine is of importance in
relation to the formation of uric acid
stones
 AMINO ACIDS
• Filtered by the glomerulus
• Reabsorbed by the proximal convoluted
tubules
LIVER FUNCTION TESTS
 FUNCTIONS OF THE LIVER
• Main site of intermediary metabolism
• Conjugation and detoxification of toxic
substances
• Storage of glycogen
• Site of immune mechanisms
• Synthesis of blood clotting factors
• AST, ALT, and alkaline phosphatase
have already been discussed and are
measures of liver function
 SERUM ALBUMIN
• Synthesized by the liver and represents
the serum protein present in greatest
quantity
• An index of the severity of chronic liver
disease
• Normal value: 3.4-4.7 g/dL
• Together with globulin is used to
measure total plasma protein (6-8 g/dL)
 BILIRUBIN
• Produced as a degradation product of heme
• Represents a balance between bilirubin
production and excretion
• Increased levels may be due to increased
production or impaired excretion
• Normal values:Direct (B2) 0.1-0.4 mg/dL
Indirect (B1) 0.2-0.7 mg/dL
Total 0.1-1.2 mg/dL
 BILIRUBIN
• Elevated bilirubin occurs in liver
disease, biliary obstruction, or
hemolysis
• Only conjugated bilirubin is seen in
urine because it is water-soluble
• Review how bilirubin is synthesized and
excreted from your previous lecture
 BILE ACIDS
• Catabolic products of cholesterol
formed in the liver
• Acids are conjugated to either glycine or
taurine to form bile salts
• Bile salts are excreted in the bile and
recirculate back to the liver through the
enterohepatic circulation
 FUNCTION OF BILE ACIDS
• Emulsification of dietary fats
• Activation of lipases
• Absorption of lipids through the
intestinal mucosa
 BILE ACIDS
• Increased levels in the serum are
caused by a decrease in the
conjugating mechanism with glycine
and taurine in liver cell damage and in
the impairment of the enterohepatic
circulation in biliary obstruction
 COAGULATION FACTORS
• All plasma procoagulants are
synthesized by the liver
• Synthesis of factors II, VII, IX, and X are
Vitamin K-dependent
• The adequacy of hepatic function can
be estimated by the Prothrombin time
 PROTHROMBIN TIME (PT)
• Normal: 11-15 secs
• Screens the extrinsic pathway
• Not affected by heparin
• Used to monitor warfarin therapy
 PARTIAL THROMBOPLASTIN
TIME (PTT or aPTT)
• 25-35 secs
• Screens the intrinsic pathway and
adequacy of all coagulation factors
except factors XIII and VII
• Best test to monitor adequacy of
heparin therapy
 Prolonged venous stasis
Blocking the flow of blood with the
tourniquet with eventually lead to a
sieving effect. Small molecules, water
and ions are forced out blood vessels
and larger molecules are concentrated
• Increases Total Protein, proteins, iron
(Fe), cholesterol, bilirubin
• Decreases potassium
 Supine vs. sitting or standing
• Going from lying (supine) to upright reduces
total blood volume by about 700 ml
• The following may decrease by 5-15% in the
supine patient:
– Total protein
– Albumin
– Lipids
– Iron
– Calcium
– Enzymes
Specimens requiring special
handling
• Should be placed immediately on ice
– Lactate
– Ammonia
– Acid phosphatase
– Plasma catecholamines
 Significantly affected by
hemolysis:

• Hemolysis-rupture of red blood cell

– Can be due to improper collection
– End result is dumping cellular contents into
blood. Mild dilution effect in some analytes
• Significant increase in potassium,
magnesium, phosphorous